1. Field of the Invention
The present invention relates to methods and to analytical systems for performing fibrinogen assays using a reagent comprising ecarin in a dry chemistry or liquid chemistry format.
2. Discussion of the Background
Coagulation assays, in general, employed as clinical assays, measure the time required for the formation of a fibrin clot. Coagulation assays are principally used for screening, diagnosis, and monitoring patients receiving anticoagulant therapy.
There are many types of coagulation assays. These include: prothrombin time (PT); partial thromboplastin time (PTT); activated partial thromboplastin time (APTT); fibrinogen assay (i.e., the measurement of the concentration of clottable fibrinogen in a sample); thrombin time, also known as thrombin clotting time (TCT); activated clotting time (ACT); etc. The most frequently performed of these assays is prothrombin time.
The prothrombin time test and the activated partial thromboplastin time test are each commonly used clinical tests to determine a patient's ability to form clots. These tests, and the other tests noted above are extensively used by hospitals, clinics, and laboratories for preoperative evaluations and for anticoagulant therapy administered to cardiac patients, among other patients. These tests are each based upon time measurements, and for the most part measure what is called an end point or clotting time, which occurs when fibrinogen is polymerized to a fibrin coagulum.
The determination of the concentration of clottable fibrinogen in plasma or whole citrated blood is important for the investigation of coagulation disturbances in patients and for following (monitoring) drug therapy that affects fibrinogen. Both immunological methods and coagulation tests have been used for the determination of fibrinogen. The immunological methods display severe diagnostic disadvantages and have consequently not achieved practical importance.
In coagulation tests, the fibrinogen content is determined by the time required for coagulum (i.e. clot) formation. The most important of these methods is the method of Clauss (see Acta Haemat. (1957) 17: 237-246).
In the Clauss method, a diluted plasma, i.e., a weak fibrinogen solution, is mixed with a concentrated thrombin solution, the amount of thrombin being about 550 U ml.sup.-1 of plasma. With the help of a calibration curve, the fibrinogen content of the sample is correlated to the time taken for the visible appearance of a coagulum. Coagulation tests in which one records photometrically the formation of turbidity during the course of coagulation are also known. See, e.g., Ratge et al, Clin. Chem. (1987) 33 (3): 420.
Finally, quantitative methods are also known in which the coagulum formed is isolated and its protein content determined. In this approach, the sample is reacted with thrombin and the coagulum formed isolated, washed and then dried. The protein content of the coagulum or its weight is then determined.
Becker et al (U.S. Pat. No. 4,692,406 ) disclose a method for the simultaneous determination of fibrinogen and of fibrinogen fission products in plasma. This method uses a snake venom enzyme with thrombin-like activity. In this method, the period of time between the addition of the enzyme and commencement of turbidity formation, which is a measure of the amount of fibrinogen fission products, is measured. The speed of turbidity formation is subsequently measured to determine the amount of fibrinogen present in the sample.
Many of these coagulation assays monitor change in sample optical density to measure the reaction. See, for example, Natelson et al (Am. J Clin. Path. (1974) 61(6): 828-833), Lipscomb (U.S. Pat. No. 4,720,787), Saito et al (U.S. Pat. No. 4,217,107), Baughman et al (U.S. Pat. No. 4,289,498), Gross et al (U.S. Pat. No. 3,458,287), Eichelberger et al (U.S. Pat. No. 4,047,890), Becker et al (U.S. Pat. No. 4,692,406), Callahan et al, "Semiquantitative Fibrinogen Determination From the PT Clotting Reaction", Tech. Bulletin Tech. THR8804, copyright 1988 by Organon Teknika, Durham, N.C., U.S.A., and Carroll et al "The Clot Signature and New Aspects in Coagulation Testing" July 1989, Ortho Diagnostic Systems Inc, Raritan, N.J., U.S.A.
In addition to being assayed by the coagulation rate method as in the Clauss method noted above, fibrinogen can be assayed by the coagulation rate as in the Clauss method modified by Vermylen et al (Clin. Chem. Acta (1963) 8:418-424), or by sulfite precipitation, Rampling et al (Clin. Chem. Acta (1976) 67:43), or by the total coagulable fibrinogen method of Ratnoff et al (J. Lab. Clin. Med. (1951) 37:316-320), or by an assay system based on the turbidity rate measurement of the conversion of fibrinogen to fibrin polymer sold by Du Pont (Du Pont Aca.TM., Du Pont Clinical Systems, Wilmington, Del. U.S.A.). The Vermylen et al method uses a glass hook or platinum loop which is continuously moved in and out of the clotting mixture until the appearance of a fibrin web indicating the end-point.